Project Summary/Abstract Aging-related adverse remodeling in cardiac and pulmonary tissues is increasingly recognized in patients with heart failure with preserved ejection fraction (HFpEF). HFpEF is associated with the development of fibrosis and decreased compliance (increased stiffness) of these vital structures. In turn, this structural rigidity results in increased work load and eventual HF with poor clinical outcomes. Little is known about the basic mechanism by which this tissue stiffness occurs. We have found evidence for elevation of expression and activity of tissue transglutaminase (TG2) in age-dependent pathologies including senescence and chronic cardiac pressure overload, occurring with enhanced glycolytic metabolism. Since TG2 is known to participate in protein cross-linking and stimulation of fibrogenesis, we hypothesize that this aging-mediated enhancement of TG2 through glycolytic stress sets in motion a variety of biologic events, including fibroblast activation and extracellular matrix protein collagen deposition and cross-linking that lead to development of tissue stiffness in experimental HFpEF. We wish to explore this novel hypothesis with the idea that intervention of TG2 activity may provide a new therapeutic approach for age-related HFpEF. In Specific Aim 1, we will utilize senescent and control fibroblasts in vitro to directly explore the influence of aging-related biologic pathways on mechanistic relationships between glycolysis, TG2, lysyl oxidase (LOX), matrix protein alteration and cell signaling and physiologic function. Expected outcome: These in vitro studies will advance our understanding about upstream aging-related mechanisms that mediate TG2 induction and associated downstream signaling pathways. In Specific Aim 2, we will use wild-type mice and a senescent-accelerated mouse (SAM) model including prone (SAMP8) and resistant (SAMR1) strains with or without pressure-overload of the cardiac left ventricle (constriction of the aorta) to assess aging-related in vivo molecular and physiologic responses of cardiac and pulmonary tissue remodeling, fibrosis and stiffness. Additionally, we will use these senescent mouse models to assess the influence of ERW1041E, a TG2 inhibitor, on these pathophysiological responses to test validation of TG2 as a significant mediator of aging-dependent tissue fibrosis and HFpEF. We will determine the extent of TG2-mediated collagen accumulation and LOX-mediated collagen oxidation in cardiac and pulmonary tissues in these mice with use of novel in vivo imaging techniques and further relate them to fibrogenic progression and remission. Further, TG2-mediated matrix protein crosslinking will be assessed using a non-invasive bioassay. Expected outcome: We hope that these studies will provide new insights into mechanisms by which tissue fibrosis and stiffness occur in aging hearts and lungs. Significance: The results of our studies may lead to similar techniques in humans to assess the presence of early stages of aging- associated HFpEF and effectiveness of related therapy directed toward inhibition of tissue fibrosis.